Mitochondria are abundant and critically important subcellular organelles whose organization and ultrastructure are complex but unexplained. This multi-PI multiscale modeling proposal seeks to use a combination of mathematical modeling and biological experiments to test the overall hypothesis that the nanoscopic ultrastructure of cardiac mitochondria accounts for the ultimate success of mitochondrial function. Mitochondria in rat cardiac ventricular myocytes will be used in the planned modeling and biological experiments. Our preliminary experiments and published data indicate that the planned work is feasible and that the three PIs can succeed in this ambitious and challenging proposal. Freshly isolated rat cardiac ventricular myocytes and those in short-term (1-3 days) culture will be prepared in the Lederer lab and imaged with electron microscope (EM) tomography by the Mannella team and analyzed quantitatively and interactively by the three PIs. Living cells will be examined by the Lederer team using confocal and super-resolution Stochastic Optical Reconstruction Microscopy (STORM). The quantitative spatial and functional data obtained from EM tomography and live cell imaging will be used to inform and constrain the multi-scale 3D modeling of mitochondria centered in the Jafri lab. The planned iterative approach to biological experiments and mathematical modeling will enable this investigation to define the structural basis for mitochondrial function for the first time. Finally, the proposed investigation seeks to include modeling of mitochondria under control conditions, when mitochondria are stressed by simple interventions and when mitochondria are altered by pressure-overload heart failure. This work therefore will not only provide fundamental new information on how mitochondria function but will also lay the foundation for novel therapies in mitochondrially involved diseases.